UUnniivveerrssiittyy ooff TTeennnneesssseeee,, KKnnooxxvviillllee TTRRAACCEE:: TTeennnneesssseeee RReesseeaarrcchh aanndd CCrreeaattiivvee EExxcchhaannggee Doctoral Dissertations Graduate School 8-2016 MMeettaabboolloommiiccss aapppprrooaacchheess ttoo ddeecciipphheerr tthhee aannttiibbaacctteerriiaall mmeecchhaanniissmmss ooff yyeerrbbaa mmaattee ((IIlleexx ppaarraagguuaarriieennssiiss)) aaggaaiinnsstt SSttaapphhyyllooccooccccuuss aauurreeuuss aanndd SSaallmmoonneellllaa eenntteerriiccaa sseerroovvaarr TTyypphhiimmuurriiuumm Caroline Sue Rempe University of Tennessee, Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Bioinformatics Commons, Chemistry Commons, and the Microbiology Commons RReeccoommmmeennddeedd CCiittaattiioonn Rempe, Caroline Sue, "Metabolomics approaches to decipher the antibacterial mechanisms of yerba mate (Ilex paraguariensis) against Staphylococcus aureus and Salmonella enterica serovar Typhimurium. " PhD diss., University of Tennessee, 2016. https://trace.tennessee.edu/utk_graddiss/3957 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Caroline Sue Rempe entitled "Metabolomics approaches to decipher the antibacterial mechanisms of yerba mate (Ilex paraguariensis) against Staphylococcus aureus and Salmonella enterica serovar Typhimurium." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. C. Neal Stewart, Major Professor We have read this dissertation and recommend its acceptance: Timothy J. Tschaplinski, Alison Buchan, Cynthia B. Peterson Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) Metabolomics approaches to decipher the antibacterial mechanisms of yerba mate (Ilex paraguariensis) against Staphylococcus aureus and Salmonella enterica serovar Typhimurium A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Caroline Sue Rempe August 2016 ii ACKNOWLEDGEMENTS I am very grateful for everyone who has helped me during my journey as a graduate student, starting with the support and encouragement of Dr. Harry Richards and Dr. Cynthia Peterson who brought me to the University of Tennessee through the SCALE-IT fellowship program. The interdisciplinary SCALE-IT program encouraged me to learn computational skills, presented me with valuable networking opportunities, and allowed me to develop collaborative projects with my peers. I also want to thank Dr. Albrecht von Arnim for his continued support and for the many opportunities I’ve had as a GST student, especially the chances to teach Perl, genetics, and a KIDSU summer camp and the opportunity to participate in the GST recruitment process. I would like to thank my advisor Dr. C. Neal Stewart for his constant support and encouragement and my committee members, Dr. Timothy Tschaplinski, Dr. Alison Buchan, and Dr. Cynthia Peterson, for their support and helpful suggestions. I additionally want to express my gratitude to Dr. Kellie Burris for fostering my microbiology skills, listening to all my ideas and problems and advising me in the directions to take my work. My gratitude also goes to Dr. Scott Lenaghan for his patient guidance and helpful discussions about my experimental successes, failures, and interpretations. I am also grateful to Dr. Timothy Tschaplinski and Nancy Engle for their willingness to work with me to learn how to run and analyze GC-MS experiments on their equipment. I would also like to thank Dr. Hayriye Bozkurt for helpful suggestions and encouragement, Dr. P. M. Davidson for the generous use of his BSL-2 lab space, and Dr. Arnold Saxton for his quick responses to my statistics questions. Additional thanks go to Dr. Shawn Campagna and his group, particularly Dr. Hector Castro Gonzalez and Eric Tague, for extracting my samples and running LC-MS analyses. Finally, I want to thank all the other individuals in my lab, GST, SCALE-IT, my family, and my friends for their continued support and encouragement. iii ABSTRACT The increasing prevalence of drug-resistant pathogens is an urgent problem that requires novel methods of bacterial control. Plant extracts inhibit bacterial pathogens and could contain antibacterial compounds with novel mechanisms of action. Yerba mate, a common South American beverage made from Ilex paraguariensis, has antibiotic activity against a broad range of bacterial pathogens. In this work, an attempt was first made to characterize the antibacterial source of an aqueous yerba mate extract by generating a series of extract fractions, collecting GC-MS and antibacterial activity profiles, and then ranking the hundreds of compounds by their presence in fractions with high antibacterial activity. Quinic acid, quercetin, and 5-hydroxy pipecolic acid were highly ranked, suggesting an association between the antibacterial activity of yerba mate against methicillin-resistant Staphylococcus aureus (MRSA). Next, metabolites that accumulated in the supernatants of Salmonella Typhimurium and Lactobacillus casei cultures were surveyed for decreases in phenolic compounds that might signify metabolism of bioactive yerba mate components. No decreases in phenolic compounds were observed. The hypothesis that phenolic compounds might chelate iron as a mechanism of antibacterial activity was also tested; exogenous iron sulfate stimulated the partial recovery of S. Typhimurium to the inhibitory effect of yerba mate in a milk system. Finally, an assessment of potential antibacterial mechanisms of action was undertaken by surveying the metabolites produced by Salmonella Typhimurium in the presence of yerba mate extract and conducting assays to assess cell membrane integrity and catalase activity. No effect on the cell membrane was observed while catalase activity was reduced in the presence of yerba mate extract. Metabolomics revealed significant differences in central carbon metabolism, the cell wall precursor UDP-N- acetylglucosamine, the regulatory metabolites alpha-ketoglutarate and acetylphosphate, the iv energy metabolite NAD+, and a match to yohimbine, which has known antibacterial activity. Future work can move closer to understanding the antibacterial value of yerba mate extract and its constituents by testing specific mechanistic hypotheses based on metabolic alterations, further examining 5-hydroxy pipecolic acid and yohimbine for antibacterial activity and mechanism, and annotating currently unknown compounds that could have antibacterial activity or be additional key metabolites pointing to specific mechanisms of action. v TABLE OF CONTENTS CHAPTER I Introduction .............................................................................................................. 1 Emergence of antibiotics............................................................................................................. 1 Antibacterial resistance overview ............................................................................................... 2 Plants as sources of novel antibacterial compounds ................................................................... 4 Scope of dissertation ................................................................................................................... 6 References ................................................................................................................................... 7 Appendix ..................................................................................................................................... 9 Figures..................................................................................................................................... 9 CHAPTER II Systems biology approaches to unraveling antibacterial mechanisms of plant phenolic compounds ..................................................................................................................... 10 Summary ................................................................................................................................... 11 Introduction ............................................................................................................................... 11 Membrane disruption by phenolic compounds ......................................................................... 13 Non-membrane mechanisms of action ...................................................................................... 16 Key functional characteristics ................................................................................................... 18 Synergistic antibacterial activity ............................................................................................... 20 Systems biology methods ......................................................................................................... 21 Conclusion ................................................................................................................................ 25 References ................................................................................................................................. 27 Appendix ................................................................................................................................... 32 Figures................................................................................................................................... 32 Tables .................................................................................................................................... 33 CHAPTER III Computational ranking of yerba mate small molecules based on their predicted contribution to antibacterial activity against methicillin-resistant Staphylococcus aureus .......... 34 Abstract ..................................................................................................................................... 35 Introduction ............................................................................................................................... 36 Materials and methods .............................................................................................................. 37 Yerba mate extractions ......................................................................................................... 37 Antimicrobial susceptibility tests .......................................................................................... 38 GC-MS sample preparation and instrument parameters ....................................................... 40 Data analysis ......................................................................................................................... 41 Results ....................................................................................................................................... 45 Discussion ................................................................................................................................. 49 Conclusion ................................................................................................................................ 53 Acknowledgements ................................................................................................................... 53 References ................................................................................................................................. 55 Appendix ................................................................................................................................... 58 Figures................................................................................................................................... 58 Tables .................................................................................................................................... 61 Supporting Information ......................................................................................................... 64 CHAPTER IV Metabolites accumulated during Salmonella enterica serovar Typhimurium and Lactobacillus casei yerba mate treatment and the rescuing effect of exogenous iron sulfate on S. Typhimurium ................................................................................................................................ 71 Abstract ..................................................................................................................................... 72 vi Introduction ............................................................................................................................... 73 Materials and methods .............................................................................................................. 75 Yerba mate aqueous extraction ............................................................................................. 75 Phenolic content determination............................................................................................. 75 Bacterial culture maintenance ............................................................................................... 76 Bacterial growth experiments ............................................................................................... 76 Metabolomics ........................................................................................................................ 77 Data analysis ......................................................................................................................... 78 Iron assays ............................................................................................................................. 79 Results ....................................................................................................................................... 81 Extract and phenolic content ................................................................................................. 81 Bacterial growth with yerba mate extract ............................................................................. 81 Metabolomics ........................................................................................................................ 82 Iron binding assays ............................................................................................................... 82 Bacterial growth with iron supplementation ......................................................................... 83 Discussion ................................................................................................................................. 83 Conclusion ................................................................................................................................ 87 Acknowledgements ................................................................................................................... 87 References ................................................................................................................................. 88 Appendix ................................................................................................................................... 91 Figures................................................................................................................................... 91 Tables .................................................................................................................................... 94 Supporting information ......................................................................................................... 95 CHAPTER V Metabolic analysis of the mechanisms of action of yerba mate extract on Salmonella enterica serovar Typhimurium .................................................................................. 97 Abstract ..................................................................................................................................... 98 Introduction ............................................................................................................................... 99 Results ..................................................................................................................................... 101 Discussion ............................................................................................................................... 103 Influx from tea extract ........................................................................................................ 107 General stress conditions .................................................................................................... 107 Antibiotic specific stressors ................................................................................................ 108 Conclusion .............................................................................................................................. 113 Materials and methods ............................................................................................................ 114 Yerba mate aqueous extraction ........................................................................................... 114 Bacterial culture maintenance ............................................................................................. 115 Bacterial growth experiments ............................................................................................. 115 Crystal violet membrane integrity assay: ............................................................................ 116 Catalase activity assay ........................................................................................................ 117 Metabolomics ...................................................................................................................... 117 Data analysis ....................................................................................................................... 118 Metabolomics data analysis ................................................................................................ 118 Acknowledgements ................................................................................................................. 120 References ............................................................................................................................... 121 Appendix ................................................................................................................................. 127 Figures................................................................................................................................. 127 vii Tables .................................................................................................................................. 132 Supporting information ....................................................................................................... 139 CHAPTER VI Conclusion ......................................................................................................... 145 VITA ........................................................................................................................................... 147 viii LIST OF TABLES Table 1. Known antibacterial mechanisms of action of phenolic compounds. (attachment) ....... 33 Table 2. Top 20 unique retention times ranked by antimicrobial significance against MRSA using random forests. ............................................................................................................ 61 Table 3. Rank biased overlap comparison of lists. ....................................................................... 62 Table 4. Top ten results for each attribute ranking method. (attachment) .................................... 63 Table 5. Classification accuracy of a single major mz peak for each of the 3 identified compounds of interest. .......................................................................................................... 63 Table 6. Predicted functional groups from Golm ......................................................................... 63 Table 7. L. casei metabolites found to be significantly different between treatments with ‘control_no_bacteria’ = milk+tea, ‘control_no_tea’ = milk+bacteria, and ‘tea’ = milk+tea+bacteria. Extracted ion intensity values normalized to a gallic acid internal standard are listed with data ranges in parentheses. (attached) ............................................ 94 Table 8. S. Typhimurium metabolites found to be significantly different between treatments with ‘control_no_bacteria= milk+tea, ‘control_no_tea ’ = milk+bacteria, and ‘tea’ = milk+tea+bacteria. Extracted ion intensity values normalized to a gallic acid internal standard are listed with data ranges in parentheses. (attached) ............................................ 94 Table 9. Significant differences between control and tea at 0 min ...................................... 132 no_tea Table 10. Significant differences between control and tea at 40 min .................................. 133 no_tea Table 11. Significant differences between control and tea at 240 min ................................ 135 no_tea Table 12. Significant differences between control and tea at 0 min .............................. 136 no_bacteria Table 13. Significant differences between control and tea at 40 min ............................ 137 no_bacteria Table 14. Significant differences between control and tea at 240 min ........................... 138 no bacteria Table S 1. Final parameters implemented with XCMS for feature detection and retention time correction of mass spectral data. ........................................................................................... 65 Table S 2. Ranking values of top 10 attributes for each ranking method. (attachment) ............... 66 Table S 3. Antimicrobial activity assays of aqueous yerba mate acetonitrile fractions. .............. 66 Table S 4. Antimicrobial activity assays of aqueous yerba mate methanol fractions. .................. 67 Table S 5. Summary of compounds identified as potential antibacterials from GC-MS data and MIC concentrations against methicillin-sensitive Staphylococcus aureus (SA) and methicillin-resistant S. aureus (MRSA) from literature. ...................................................... 68 Table S 6. Accuracies of classification methods shown in confusion matrices. ........................... 68 Table S 7. Predicted functional groups listed for the top 10 compounds with retention time (RT), retention index (RI) and name if known. .............................................................................. 69 Table S 10. Peak-picking parameters for metabolomics data analysis with Maven software. These parameters were used for both targeted and untargeted analyses but targeted analyses were limited to identifiable compounds. ............................................................................. 143 Table S 11. Pure catalase tested for activity in the presence of yerba mate extract (control no bacteria ). Pure catalase (5 U) in buffer was compared to pure catalase (5 U) with incubated yerba mate extract (control ). No significant difference was observed between catalase in no bacteria buffer and catalase treated with incubated yerba mate extract sampled at different times. 144 Table S 12. Complete list of all metabolites found with LC-MS. (attachment) ......................... 144 Table S 13. List of all identified metabolites found with LC-MS. (attachment) ........................ 144